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. 1994 Jan;60(1):26–30. doi: 10.1128/aem.60.1.26-30.1994

Electrophoretic Transfer from Polyacrylamide Gel to Nitrocellulose Sheets, a New Method To Characterize Multilocus Enzyme Genotypes of Klebsiella Strains

Marie-Laure Combe 1, Jean-Louis Pons 1,*, Richard Sesboue 1, Jean-Pierre Martin 1
PMCID: PMC201264  PMID: 16349155

Abstract

A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-μl samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.

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Selected References

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  1. Boerlin P., Rocourt J., Piffaretti J. C. Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis. Int J Syst Bacteriol. 1991 Jan;41(1):59–64. doi: 10.1099/00207713-41-1-59. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Caugant D. A., Bøvre K., Gaustad P., Bryn K., Holten E., Høiby E. A., Frøholm L. O. Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers. J Gen Microbiol. 1986 Mar;132(3):641–652. doi: 10.1099/00221287-132-3-641. [DOI] [PubMed] [Google Scholar]
  4. Edmondson A. S., Cooke E. M. The development and assessment of a bacteriocin typing method for Klebsiella. J Hyg (Lond) 1979 Apr;82(2):207–223. doi: 10.1017/s0022172400025626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gaston M. A., Warner M. Electrophoretic typing of Enterobacter cloacae with a limited set of enzyme stains. Epidemiol Infect. 1989 Oct;103(2):255–264. doi: 10.1017/s0950268800030612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Goullet P. Distinctive electrophoretic patterns of esterases from Klebsiella pneumoniae, K. oxytoca, Enterobacter aerogenes and E. gergoviae. J Gen Microbiol. 1980 Apr;117(2):483–491. doi: 10.1099/00221287-117-2-483. [DOI] [PubMed] [Google Scholar]
  7. Goullet P., Picard B. Characterization of enterobacteria by esterase specific-activity profiles. J Gen Microbiol. 1990 Mar;136(3):431–440. doi: 10.1099/00221287-136-3-431. [DOI] [PubMed] [Google Scholar]
  8. Lehmann P. F., Hsiao C. B., Salkin I. F. Protein and enzyme electrophoresis profiles of selected Candida species. J Clin Microbiol. 1989 Mar;27(3):400–404. doi: 10.1128/jcm.27.3.400-404.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. McLellan T., Ramshaw J. A. Serial electrophoretic transfers: a technique for the identification of numerous enzymes from single polyacrylamide gels. Biochem Genet. 1981 Aug;19(7-8):647–654. doi: 10.1007/BF00483998. [DOI] [PubMed] [Google Scholar]
  10. Musser J. M., Granoff D. M., Pattison P. E., Selander R. K. A population genetic framework for the study of invasive diseases caused by serotype b strains of Haemophilus influenzae. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5078–5082. doi: 10.1073/pnas.82.15.5078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ochman H., Selander R. K. Evidence for clonal population structure in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jan;81(1):198–201. doi: 10.1073/pnas.81.1.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Piffaretti J. C., Kressebuch H., Aeschbacher M., Bille J., Bannerman E., Musser J. M., Selander R. K., Rocourt J. Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proc Natl Acad Sci U S A. 1989 May;86(10):3818–3822. doi: 10.1073/pnas.86.10.3818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pons J. L., Picard B., Niel P., Leluan G., Goullet P. Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens. Appl Environ Microbiol. 1993 Feb;59(2):496–501. doi: 10.1128/aem.59.2.496-501.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rennie R. P., Duncan I. B. Combined biochemical and serological typing of clinical isolates of Klebsiella. Appl Microbiol. 1974 Oct;28(4):534–539. doi: 10.1128/am.28.4.534-539.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rennie R. P., Nord C. E., Sjoberg L., Duncan I. B. Comparison of bacteriophage typing, serotyping, and biotyping as aids in epidemiological surveillance of Klebsiella infections. J Clin Microbiol. 1978 Dec;8(6):638–642. doi: 10.1128/jcm.8.6.638-642.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Riser E., Noone P., Bonnet M. L. A new serotyping method for Klebsiella species: evaluation of the technique. J Clin Pathol. 1976 Apr;29(4):305–308. doi: 10.1136/jcp.29.4.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Selander R. K., Caugant D. A., Ochman H., Musser J. M., Gilmour M. N., Whittam T. S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol. 1986 May;51(5):873–884. doi: 10.1128/aem.51.5.873-884.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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